Abstract: Segmental long bone defect surgeries require large devitalized allograft transplantations to replace missing host bone segments; however, significant problems often arise due to the impaired ability of the devitalized allograft to incorporate into the host bone. Recently, several discoveries in our lab have provided the basis for selecting, expanding and delivering of a substantial number of mesenchymal stem cells (MSCs), which retain their original stem cell characteristics, to be utilized in skeletal therapeutic applications: i) Notch2-selected and Notch activation by recombinant Jagged1 (JAG1) ligand expanded MSCs possess enhanced potentials for chondrogenic and osteogenic differentiation; ii) MSC sheets generated in temperature-responsive culture dishes exhibit a more robust effect on allograft incorporation into the host bone when transplanted to the site of the defect as a tissue-engineered periosteum surrounding the implanted graft; iii) Notch signaling induces rapid TWIST1 expression in MSCs. Based on these novel findings, we hypothesize that cell sheets generated with JAG1 expanded Notch2 positive MSCs on thermo-responsive culture dishes will significantly enhance critical segmented bone defects healing in a femoral allograft repair mouse model. To test this hypothesis, we will first to determine whether Notch2-selected and JAG1 expanded MSC sheets significantly enhance allograft healing and incorporation using our pre-clinical femoral allograft mouse model, as compared to cell sheets derived from traditionally isolated and expanded MSCs. Second, we will examine whether TWIST1 is require for JAG1-mediated MSC proliferation and maintenance in culture. Finally, we will determine how Notch activation by JAG1 involved in MSC sheet- induced angiogenesis. Data generated by this proposal will likely 1) identify optimized cell populations for generating tissue engineered periosteum (next generation MSC sheets), 2) validate a novel therapeutic approach for enhancing the repair of massive devitalized bone allograft and lead to advances in understanding the mechanism underlying MSC sheet-induced tissue repair, and 3) pave the path for large pre-clinical animal studies and subsequent clinical trials.